Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• This disclosure relates to antibody affinity maturation of antibodies targeting an antigen or antigenic epitope using natural liability-free complementarity-determining regions (CDRs).
• CDRs complementarity-determining regions
• the quality of a monoclonal antibody is often measured by its affinity to the target antigen.
• a common step in the development of a therapeutic antibody is submitting selected leads to affinity maturation campaigns on the assumption that higher affinities will lead to higher drug potency.
• the present disclosure is based, at least in part, on an improved method of antibody affinity maturation using natural complementarity-determining regions (CDRs) that are substantially free of liabilities.
• CDRs are natural, , and derived from sequencing rearranged antibody genes. These CDRs are further screened for member sequence liabilities that potentially negatively impact developability of the assembled antibody from the expressed light chain and the expressed heavy chain carrying these CDRs. The CDRs having these liabilities are removed from consideration in assembling the antibody libraries contemplated herein. The remaining natural, liability-free CDRs are combined and assembled into antibodies and screened for improved antigen-binding affinity compared to the control, parental antibody, improvement affinity can range -picomolar binding affinity.
• an antibody library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises the following CDR sequences: a V H -CDR1 sequence, a V H -CDR2 sequence, a V H -CDR3 sequence, a V L -CDR1 sequence, a V L -CDR2 sequence, and a V L -CDR3 sequence, wherein two or more of the CDR sequences are the same for each of the plurality of antibodies (“invariant CDR sequences”); and wherein each of the remaining CDR sequences comprise a plurality of unique CDRs derived from sequences of the CDRs found in naturally occurring antibodies (“variant CDR sequences”).
• “Naturally occurring antibodies” means antibodies that do not reflect antibody engineering or other genetic engineering.
• the two or more invariant CDR sequences may be selected from the group consisting of: (i) V L -CDR3, V H -CDR1, V H -CDR2, and V H -CDR3; (ii) V L -CDR1, V L -CDR2, V H -CDR1, V H -CDR2, and V H -CDR3; (iii) V L -CDR1, V L -CDR2, V L -CDR3, and V H -CDR3; (iv) V L -CDR1, V L -CDR2, and V H -CDR3; and (v) V L -CDR1, V L -CDR2, V L -CDR3, V H -CDR1 and V H - CDR2.
• the invariant CDR sequences may be derived from a parental antibody binding to a single antigen or antigenic epitope.
• an antibody library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises the following CDR sequences: a V H -CDR1 sequence, a V H -CDR2 sequence, a V H -CDR3 sequence, a V L -CDR1 sequence, a V L -CDR2 sequence, and a V L -CDR3 sequence, wherein two or more of the CDR sequences are the same for each antibody of the plurality of antibodies (“invariant CDR sequences”); and wherein each of the remaining CDR sequences (“variant CDR sequences”), except the HCDR3, comprises a plurality of CDRs derived from the sequences of CDRs found in naturally occurring antibodies, and the HCDR3 comprises a plurality of variants of the parental HCDR3.
• the HCDR3 comprises single mutations of the parental HCDR3.
• the two or more invariant CDR sequences may be selected from the group consisting of: (i) V L- CDR3, V H -CDR1, V H -CDR2, and V H- CDR3; (ii) V L -CDR1, V l- CDR2, V H -CDR1, V H -CDR2, and V H -CDR3; (iii) V L -CDR1, V L -CDR2, V L -CDR3, and V H - CDR3; and (iv) V L -CDR1, V L -CDR2, V L -CDR3, V H -CDR1, and V H -CDR2.
• the invariant CDR sequences may be derived from a parental antibody binding to a single antigen or antigenic epitope.
• the invariant CDR sequences may comprise a combination of V H -CDR1, V H -CDR2, V H -CDR3, V L -CDR1, V L -CDR2 and/or V L -CDR3.
• the invariant and variant CDR sequences are free of one or more of the following liabilities: (i) a glycosylation site, (ii) a deamidation site, (iii) an isomerization site, (iv) unpaired cysteine, (v) net charge greater than 1, (vi) a tripeptide motif containing at least two aromatic residues, (vii) a motif that promotes aggregation, (viii) a poly specificity site, (ix) a protease sensitive site, (x) an integrin binding site, (xi) a lysine glycosylation site, (xii) a metal catalyzed fragmentation site, (xiii) a poly specificity aggregation site, and (xiv) a streptavidin binding motif.
• the glycosylation site may comprise the motif NXS, NXT, or NXC, in which X represents any naturally-occurring amino acid residue except for proline
• the deamidation site may comprise the motif of NG, NS, NT, NN, NA, NH, ND, GNF, GNY, GNT, or GNG.
• the isomerization site may comprise the motif of DT, DH, DS,
• the tripeptide may be HYF or HWH.
• the motif that promotes aggregation may comprise the motif of
• the poly specificity site may comprise the motif of GG, GGG, RR, VG, W, WV, WW, WWW, YY, or WXW, in which X represents any amino acid residue.
• the protease cleavage site may comprise the motif of DX, in which X is P, G, S, V, Y, F, Q, K, L, or D.
• the integrin binding site may comprise RGD, RYD, LDV, or
• the lysine glycosylation site may comprise KE, EK, or ED.
• the metal catalyzed fragmentation site may comprise the motif of HS, SH, KT, HXS, or SXH, in which X represents any amino acid residue.
• the poly specificity aggregation site may comprise a motif of X1X2X3, wherein each of XI, X2, and X3 independently is selected from the group consisting of F, I, L, V, W and Y.
• the streptavidin binding motif may comprise the motif HPQ, EPDW, PWXWL, in which X represents any amino acid residue, GDWVFI, or PWPWLG.
• any one of the antibody libraries disclosed above and herein may be a full-length antibody library, a Fab antibody library, a single-chain antibody library, or a single domain antibody library.
• any one of the antibody libraries disclosed above and herein may be a human antibody library.
• the present disclosure provides a method for generating an antibody library, the method comprising: (a) selecting two or more CDR sequences from the group consisting of a V H -CDR1 sequence, a V H -CDR2 sequence, a V H -CDR3 sequence, a V L -CDR1 sequence, a V L -CDR2 sequence, and a V L -CDR3 sequence; and (b) generating an antibody library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises the following CDR sequences: (i) the two or more CDR sequences selected in (a) (“invariant CDR sequences”); and (ii) a unique combination of remaining CDR sequences not selected in (a) (“variant CDR sequences”), wherein the variant CDR sequences are selected from the group consisting of a V H -CDR1 sequence, a V H -CDR2 sequence, a V H -CDR3 sequence, a V L -C
• the present disclosure also provides a method for generating an antibody library, the method comprising: (a) selecting two or more CDR sequences from the group consisting of a V H -CDR1 sequence, a V H -CDR2 sequence, a V H -CDR3 sequence, a V L - CDR1 sequence, a V L -CDR2 sequence, and a V L -CDR3 sequence; and (b) generating an antibody library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises the following CDR sequences: (i) the two or more CDR sequences selected in (a) (“invariant CDR sequences”); and (ii) a unique combination of remaining CDR sequences not selected in (a) (“variant CDR sequences”), wherein the variant CDR sequences are selected from the group consisting of a V H -CDR1 sequence, a V H -CDR2 sequence, a V H -CDR3 sequence, a V L -
• a method for identifying an antibody with improved affinity to a target antigen or a target antigenic epitope comprising: (a) generating an antibody library according to any of the method described above and herein, wherein the two or more invariant CDR sequences are derived from the parental antibody to be affinity matured; and (b) screening the antibody library to isolate an antibody that binds more tightly to the target antigen or target antigenic epitope relative to a control, thereby identifying an antibody with improved affinity.
• a method for improving the developability of an antibody that binds a target antigen or a target antigenic epitope comprising: (a) generating an antibody library according to any of the methods described above and herein, wherein the two or more invariant CDR sequences are derived from the parental antibody to be affinity matured, and wherein the invariant and variant CDR sequences are free of one or more of sequence liabilities; and (b) screening the antibody library to isolate an antibody that binds more tightly to the target antigen or the target antigenic epitope relative to a control and lacks one or more of the sequence liabilities found in the CDRs of the original antibody, thereby improving the developability of the antibody.
• the two or more invariant CDR sequences may be selected from the group consisting of: (i) V L -CDR3, V H -CDR1, V H -CDR2, and V H -CDR3; (ii) V L -CDR1, V L - CDR2, V H -CDR1, V H -CDR2, and V H -CDR3; (iii) V L -CDR1, V L -CDR2, V L -CDR3, and V H - CDR3; (iv) V L -CDR1, V L -CDR2, and V H -CDR3; and (v) V L -CDR1, V L -CDR2, V L -CDR3, V H - CDR1 and V H -CDR2.
• the invariant and variant CDR sequences are free of one or more of the following liabilities: (i) a glycosylation site, (ii) a deamidation site, (iii) an isomerization site, (iv) unpaired cysteine, (v) net charge greater than 1 , (vi) a tripeptide motif containing at least two aromatic residues, (vii) a motif that promotes aggregation, (viii) a poly specificity site, (ix) a protease sensitive site, (x) an integrin binding site, (xi) a lysine glycosylation site, (xii) a metal catalyzed fragmentation site, (xiii) a poly specificity aggregation site, and (xiv) a streptavidin binding motif.
• the glycosylation site may comprise the motif NXS, NXT, or NXC, in which X represents any naturally-occurring amino acid residue except for proline.
• the deamidation site may comprise the motif of NG, NS, NT, NN, NA, NH, ND, GNF, GNY, GNT, or GNG.
• the isomerization site may comprise the motif of DT, DH, DS,
• the tripeptide may be HYF or HWH.
• the motif that promotes aggregation may comprise the motif of
• the poly specificity site may comprise the motif of GG, GGG, RR, VG, W, WV, WW, WWW, YY, or WXW, in which X represents any amino acid residue.
• the protease cleavage site may comprise the motif of DX, in which X is P, G, S, V, Y, F, Q, K, L, or D.
• the integrin binding site may comprise RGD, RYD, LDV, or
• the lysine glycosylation site may comprise KE, EK, or ED.
• the metal catalyzed fragmentation site may comprise the motif of HS, SH, KT, HXS, or SXH, in which X represents any amino acid residue.
• the poly specificity aggregation site may comprise a motif of X1X2X3, wherein each of XI, X2, and X3 independently is selected from the group consisting of F, I, L, V, W and Y.
• the streptavidin binding motif may comprise the motif HPQ, EPDW, PWXWL, in which X represents any amino acid residue, GDWVFI, or PWPWLG.
• any one of the antibody libraries disclosed above and herein may be a full-length antibody library, a Fab antibody library, a single-chain antibody library, or a single domain antibody library.
• any one of the antibody libraries disclosed above and herein may be a human antibody library.
• any of the antibody libraries disclosed herein may be of a suitable format, e.g., a library of full-length antibodies, a library of antigen-binding fragments such as Fab fragments, a library of single-chain antibodies, or a library of single-domain antibodies (e.g., VHH antibodies).
• the antibody library disclosed herein may be a human antibody library.
• the antibody library disclosed herein may be a camelid VHH antibody library.
• the present disclosure provides a VHH library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises the following CDR sequences: a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, wherein one or more of the CDR sequences is(are) the same for each antibody of the plurality of antibodies (invariant CDR Sequence(s)); and wherein each of the remaining CDRs comprises a plurality of CDRs derived from the sequences of CDRs found in naturally occurring antibodies (variant CDRs).
• the invariant CDR sequence(s) is(are) derived from a parental VHH binding to a single antigen or antigenic epitope.
• the present disclosure provides a VHH library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises the following CDR sequences: a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, wherein one or more of the CDR sequences is(are) the same for each antibody of the plurality of antibodies (“invariant CDR Sequence(s)”); and wherein each of the remaining CDRs, except the HCDR3, comprises a plurality of CDRs derived from the sequences of CDRs found in naturally occurring antibodies (“variant CDRs”), and wherein the HCDR3 comprises a plurality of variants of the parental HCDR3.
• the HCDR3 comprises single mutations of the parental HCDR3.
• the invariant CDR sequence(s) is(are) derived from a parental VHH binding to a single antigen or antigenic epitope.
• the present disclosure provides a method for generating a VHH library, the method comprising: (a) selecting one or more CDR sequences from the group consisting of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence; and (b) generating an antibody library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises the following CDR sequences: (i) the one or more CDR sequences selected in (a) (“invariant CDR sequence(s)”); and (ii) a unique combination of remaining CDR sequences not selected in (a) (“variant CDR sequences”), wherein each of the variant CDR sequences comprises a plurality of CDRs derived from the sequences of CDRs found in naturally occurring antibodies.
• the present disclosure provides a method for generating a VHH library, the method comprising: (a) selecting one or more CDR sequences from the group consisting of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence; and (b) generating an antibody library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises the following CDR sequences: (i) the one or more CDR sequences selected in (a) (“invariant CDR sequence(s)”); and (ii) a unique combination of remaining CDR sequences not selected in (a) (“variant CDR sequences”), wherein each of the variant CDR sequences, except the HCDR3, comprise a plurality of CDRs derived from the sequences of CDRs found in naturally occurring antibodies, and wherein the HCDR3 comprises a plurality of variants of the parental HCDR3.
• the CDR3 may comprise single mutations of the parental HCDR3.
• the CDR sequences are free of one or more of the following sequence liabilities: (i) a glycosylation site, (ii) a deamidation site, (iii) an isomerization site, (iv) unpaired cysteine, (v) net charge greater than 1 , (vi) a tripeptide motif containing at least two aromatic residues, (vii) a motif that promotes aggregation, (viii) a poly specificity site; (ix) a protease sensitive site, (x) an integrin binding site, (xi) a lysine glycosylation site, (xii) a metal catalyzed fragmentation site, (xiii) a poly specificity aggregation site; and (xiv) a streptavidin binding motif.
• the glycosylation site may comprise the motif NXS, NXT, or NXC, in which X represents any naturally-occurring amino acid residue except for proline;
• the deamidation site may comprise the motif of NG, NS, NT, NN, NA, NFI, ND, GNF, GNY, GNT, or GNG;
• the isomerization site may comprise the motif of DT, DH, DS, DG, or DD; tripeptide is FIYF or HWH;
• the motif that promotes aggregation may comprise the motif of FHW;
• the poly specificity site may comprise the motif GG, GGG, RR, VG, W, WV, WW, WWW, YY, or WXW, in which X represents any amino acid residue;
• the protease cleavage site may comprise the motif of DX, in which X is P, G, S, V, Y, F, Q, K, L, or D;
• FIG. 1A shows the schematic representation of the parental antibody scFv and the three libraries produced in phase 1: L1L2, L3, and H1H2. Each library has diversity introduced in the indicated CDRs. These phase 1 libraries allow for screening and selecting a pool of the best affinities for the respective tested CDRs, the pool of best affinity CDRs are then to be later used in the phase 2 for generating combinatorial libraries having at least three CDRs replaced and three natural non-replaced CDRs.
• FIG. IB shows the sensorgram of the parental antibody binding to the antigen generated by surface-plasmon resonance in increasing antigen concentrations (0.16 nM to 100 nM with 5-fold increase in each step).
• FIG. 1C shows the schematic for creating a L1L2 library in phase 1; this is an exemplary for also creating a L3, and a HIH2 libraries.
• An LI sub-library pool is generated by PCR using specific primers as described herein (the LCDR1 pool).
• an L2 sub-library is generated by PCR (the LCDR2 pool).
• PCR with selected designed primers then allow the generation of the L1L2 library from these two sub-libraries and assembling L1L2 into the parental antibody scFv (which has unchanged LCDR3 and HCDRs) and thereby produced the L1L2 library in phase 1.
• Each library has diversity introduced in the indicated CDRs.
• FIG. 2A shows the schematic representation of yeast display selections using equilibrium and kinetic protocols.
• FIG. 2B shows the outline of the selection rounds performed in phase 1 with the three phase 1 libraries (L1L2, L3, H1H2).
• FIG. 2C shows the yeast display binding profiles of the parental scFv and the three phase 1 libraries at increasing antigen concentration assessed by flow cytometry. Binding to antigen (APC fluorescence) is shown on the Y axis and scFv display (PE fluorescence) is shown on the X axis.
• FIG. 2D shows the yeast display binding profile of the parental scFv and the libraries after 5 rounds of selection. A gate representing the parental population is shown in all plots for comparison. In the last two columns, after labeled antigen incubation, cells were washed and incubated with unlabeled antigen for 2h and 4h to evaluate the stability of binding.
• FIG. 3A shows the schematic representation of parental antibody scFv and the two Combo libraries produced: Combo 1 has diversity in LCDRl-3 and HCDRl-2 and Combo 2 has diversity in LCDR3 and HCDRl-2.
• FIG. 3B shows the outline of the selection rounds performed in phase 2 with the two Combo libraries.
• FIG. 3C shows the yeast display binding profile of the combo libraries before any rounds of selection were performed. Binding to antigen (APC fluorescence) is shown on the Y axis and scFv display (PE fluorescence) is shown on the X axis.
• FIG. 3D shows the yeast display binding profile of the parental scFv and the combo libraries after 3 rounds of selection.
• FIG. 4A shows the Venn diagram representing the number of unique clones identified by Sanger sequencing coming from Combo libraries 1 and 2.
• FIG. 4B shows the chord diagram showing the connections between each of the CDRs identified.
• FIG. 4C shows the sequence logos comparing the CDRs from the parental scFv, designed for the library, and observed in the sequenced clones. Letter heights indicate the frequency of the given amino acid and letter width represent frequency of non-gap at the position. Dashed squared show regions that converged for an amino acid different from the parental.
• FIG. 4D shows the heat map showing the number of amino acid changes from parental in each CDR and the total number per clone. Each row represents an identified clone. Clones are ordered from least to most total changes.
• FIG. 4A shows the Venn diagram representing the number of unique clones identified by Sanger sequencing coming from Combo libraries 1 and 2.
• FIG. 4B shows the chord diagram showing the connections between each of the CDRs identified.
• FIG. 4C shows the
• FIG. 4E shows the histograms representing the number of amino acid changes in each CDR identified and the total number of changes per clone.
• FIG. 4F is a histogram showing the total edit distance for each selected clone compared to the parental clone. The distance is calculated individually for each CDR and then summed to find the total distance
• FIG. 5A shows the antigen binding to yeast displaying the parental scFv when competing with 23 affinity matured clones (A01 to B 12) and an unrelated scFv-Fc (control). All values are shown normalized by the control.
• FIG. 5B shows shows the observed on- (ka) and off-rates (kd) for the parental and identified clones. Isoaffinity curves are shown as dashed diagonal lines.
• FIG. 5C shows the SPR sensorgrams for 23 of the identified clones shown in duplicate (side-by- side). Name of the clone and calculated KD is shown in each plot. Measurements where the calculated off-rate (kd) is estimated to be less than 10 5 s 1 are outlined in black.
• FIG. 6 shows the flow cytometric analysis of antigen binding to the yeast displaying the parental scFv when competing with 23 affinity matured clones (A01 to B12) and an unrelated scFv-Fc (control). The scFv-Fc supernatants were incubated with the labeled antigen (10 nM) for 15 minutes, then yeast cells displaying the parental molecule were added, followed by fluorescent staining to detect binding to the antigen.
• FIG. 7A shows the schematic representation of the parental antibody scFv and the four libraries produced in phase 1: H1H2, H3, F1F2 and L3. Each library has diversity introduced in the indicated CDRs. These phase 1 libraries allow for screening and selecting a pool of the best affinities for the respective tested CDRs, the pool of best affinity CDRs are then to be later used in the phase 2 for generating combinatorial libraries having the six CDRs replaced.
• FIG. 7B shows the schematic for creating a H1H2 library in phase 1; this is an exemplary for also creating a H3, F1L2 and F3 libraries. An HI sub-library pool is generated by PCR using specific primers as described herein (the HCDR1 pool).
• FIG. 8A shows the yeast display binding profiles of the four phase 1 libraries at increasing antigen concentration assessed by flow cytometry. Binding to antigen (APC fluorescence) is shown on the Y axis and scFv display (PE fluorescence) is shown on the X axis.
• FIG. 8B shows the outline of the selection rounds performed in phase 1 with the four phase 1 libraries (H1H2, H3, L1L2 and L3).
• FIG. 8C shows the yeast display binding profile of the four phase 1 libraries (H1H2, H3, L1L2 and L3) after 3 rounds of selection.
• FIG. 9A shows the schematic representation of parental antibody scFv and the Combo libraries produced: Combo 1 has diversity in LCDRl-3 and FICDRl-3, while the HCDR3 for Combo 2 remains constant.
• FIG. 9B shows the outline of the four selection rounds performed in phase 2 with the two Combo libraries.
• FIG. 9C shows the yeast display binding profile of the Combo libraries before any rounds of selection were performed. Binding to antigen (APC fluorescence) is shown on the Y axis and scFv display (PE fluorescence) is shown on the X axis.
• FIG. 9D shows the yeast display binding profile of the parental scFv and the combo libraries after four rounds of selection, at 1 nM and 1 mM of antigen.
• FIG. E compares the yeast display binding profile of the parental scFv and the combo libraries after four rounds of selection, when stained with 750 nM of antigen, and at different release time points.
• FIG. 10A shows the Venn diagram representing the number of unique clones identified by Sanger sequencing from Combo libraries 1 and 2, derived from the four phase 1 libraries.
• FIG. 10B shows the heat map showing the number of amino acid changes from parental in each CDR and the total number per clone. Each row represents an identified clone. Clones are ordered from least to most total changes.
• FIG. IOC shows the histograms representing the number of amino acid changes in each CDR identified and the total number of changes per clone.
• FIG. 10D shows the sequence logos comparing the CDRs from the parental scFv, designed for the library, and observed in the sequenced clones. Letter heights indicate the frequency of the given amino acid and letter width represent frequency of non-gap at the position. Mutational frequencies of the HCDR3 were too low to be visualized in the sequence logo.
• Affinity maturation is a necessary step for the development of potent therapeutic molecules from a library of naive antibodies targeting and binding an antigen or an antigenic epitope.
• the quality of a monoclonal antibody is often measured by its affinity to the target antigen or the target antigenic epitope.
• Flence a common step in the development of a therapeutic antibody is submitting selected leads to affinity maturation campaigns on the assumption that higher affinities will lead to higher drug potency.
• Many techniques are used for antibody affinity maturation, including error-prone PCR, chain shuffling, targeted CDR mutation, and others. These are effective but can negatively affect antibody stability or alter epitope recognition.
• sequence liabilities such as glycosylation, asparagine deamidation, aspartate isomerization, aggregation motifs, and others that potentially could affect the developability of the lead antibody arising from the naive library targeting an antigen. All these can potentially create the need for new rounds of engineering or even abolish the usefulness of the antibody as a therapeutic molecule.
• the present disclosure provides improved methods of antibody affinity maturation using natural complementarity-determining regions (CDRs) that are substantially free of sequence liabilities as well as antibody libraries so produced.
• CDRs complementarity-determining regions
• the antibody libraries of the present disclosure are functionally much larger than libraries of similar genetic size, in which antibodies are present that contain any of the sequence liabilities.
• the antibody libraries disclosed herein have a much larger effective diversity.
• the term “liability” refers to a motif in an antibody that would negatively affect one or more desired features of the antibody (e.g., stability, good expression in an expression or display system, proper folding, no or reduced aggregation, solubility, no or reduced integrin binding, no or reduced glycosylation, no or reduced deamidation, no or reduced isomerization, no unpaired cysteine, or no or reduced protease sensitivity, etc.).
• the present disclosure demonstrated the possibility of performing affinity maturation of a low-nanomolar affinity antibody to the low-picomolar range by a) replacing all CDRs, except HCDR3, with a collection of known human CDRs; and b) also replacing the HCDR3 with limited variants. More specifically, provided herein is a method for generating a combinatorial antibody library with improved epitope-binding affinity, improvement in the order 10 3 , by switching out one or more CDRs from a collection of known CDRs obtained from a naive naturally occurring antibody library, and for limited variants in HCDR3.
• a single lead antibody from this naive naturally occurring antibody library is selected as the parent antibody which would provide VH and VL scaffold framework for replacing the CDRs described below.
• That lead antibody of lower affinity has three V H -CDRS and three V L -CDRS as its “natural” or parental CDRs.
• the assortments of natural CDRs are mixed and assembled into a respective VH or VL such that the arrangements of all the V H -CDRSI-3 and V L - CDRsl-3 are not as they are in the naturally occurring antibody library, i.e., artificial combinations of the natural CDRs.
• developerability encompasses the feasibility of molecules to successfully progress from discovery to development via evaluation of their physicochemical properties.
• the disclosure provides an antibody library comprising a plurality of antibodies, wherein each antibody therein comprises: a V H -CDR1 sequence, a V H -CDR2 sequence, a V H -CDR3 sequence, a V L -CDR1 sequence, a V L -CDR2 sequence, and a V L -CDR3 sequence; wherein one or more CDR sequences selected from the group consisting of a V H - CDR1 sequence, a V H -CDR2 sequence, a V H -CDR3 sequence, a V L -CDR1 sequence, a V L - CDR2 sequence, and a V L -CDR3 sequence, are the same for each antibody of the plurality of antibodies; and each of the remaining non-selected CDRs making up the V H and V L is derived from the sequences of CDRs found in naturally occurring antibodies.
• the defined collection of natural CDRs from a known naive antibody library is purged of sequence liabilities and the reminding liability-free CDRs were inserted these into the lead antibody molecule (parental, not altered, see, FIG. 1A and FIG. 3A) from the original naturally occurring antibody library in one or two sites at a time (LCDRl-2, LCDR3, HCDRl-2) while HCDR3 and framework regions remained constant throughout the process.
• various LCDRl-2 would be inserted into the lead antibody while the LCDR3/ HCDRl-3 remain constant.
• a combinatorial antibody library is generated with variable LCDRl-2 and the same LCDR3/ HCDRl-3 (see, FIG. 1A, the L1L2 library).
• various LCDR3 are used to insert into the lead antibody where the LCDRl/2 and HCDRl-3 remain constant.
• a combinatorial antibody library is generated with variable LCDR3 and the same LCDRl-2/ HCDRl-3 (see, FIG. 1A, the L3 library).
• various HCDRl-2 may be inserted into the lead antibody while the LCDRl-3/ HCDR3 remain constant.
• a combinatorial antibody library is generated with variable HCDRl-2 and the same LCDRl-3/ HCDR3 (see, FIG. 1A, the H1H2 library).
• various LCDR3 and various HCDRl-2 may be used to insert into the lead antibody where the LCDRl/2 and HCDR3 remain constant.
• a combinatorial antibody library is generated with variable LCDR3, variable HCDRl-2 and the same LCDRl-2/ HCDR3 (see, FIG. 3A, the Combo 2 library).
• various LCDRl-3 and various HCDRl-2 may be used to insert into the lead antibody where only the HCDR3 remain constant.
• a combinatorial antibody library is generated with variable LCDRl-3, variable HCDRl-2 and the same HCDR3 (see,
• FIG. 3A the Combo 2 library
• variable LCDRl-3 and HCDRl-2 and also HCDR3 are liability free.
• the methods involve first selecting a parental or lead antibody with desired binding and/or biological characteristics. After initial identification of lead antibodies that bind to a target of interest, antibody clones may be tested for their ability to achieve a desired biological activity. Such activity may be agonistic or antagonistic. Once a lead’s desired activity is identified, the methods disclosed herein enable an antibody to be generated which maintains the epitope specificity and the desired biological activity, while increasing the affinity, which presumably will also increase potency (see, e.g., Rosenfeld et al., 2017; Hurlburt et al. 2020). Any antibody or fragment thereof may be selected for use in the present disclosure.
• An antibody is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
• antibody encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain antibody (scFv), single chain Fab (scFab), fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single domain antibodies (also known as nanobodies, e.g., a VH only antibody such as the VhH or VHH antibodies found in camelids), or multispecific antibodies (e.g., bispecific antibodies), or and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of the required specificity,, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
• antigen-binding fragments thereof such as Fab, Fab', F(ab')2, Fv
• scFv single-chain antibody
• scFab single chain Fab
• An antibody as disclosed herein includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
• immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
• the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
• the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
• a typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding.
• VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”).
• CDR complementarity determining regions
• FR framework regions
• Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• the extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Rabat definition, the Chothia definition, the AbM definition, and/or the contact definition, all of which are well known in the art. See, e.g., Rabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al, (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See, also hgmp.mrc.ac.uk and bioinf.org.uk/abs.
• the antibodies described herein may be a full-length antibody, which contains two heavy chains and two light chains, each including a variable domain and a constant domain.
• the antibodies described herein can be an antigen-binding fragment of a full-length antibody.
• binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab') 2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementar
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv).
• scFv single chain Fv
• any of the antibodies described herein can be either monoclonal or polyclonal.
• a “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.
• Single-domain antibodies also known as nanobodies, are also within the scope of the present disclosure.
• a single-domain antibody contains only a heavy chain (VHH).
• Heavy chain only antibodies (HcAb) are naturally produced by camelids and sharks.
• the antigen binding portion of the HcAb is comprised of the VHH fragment. See, e.g., Vincke et al., Methods Mol Biol. 911:15-26 (2012).
• the antibody libraries disclosed herein may contain a population of antibodies of any suitable format.
• the antibody library disclosed herein comprise a population of full-length antibodies, which may be of any suitable family (e.g., IgG, or IgA).
• the antibody library disclosed herein comprise a population of antigen-binding fragments, for example Fab fragments.
• the antibody library disclosed herein comprise a population of single-chain antibodies.
• the antibody library disclosed herein may comprise a population of single-domain antibodies such as VHH fragments.
• the term “partially diversified antibody library” refers to an antibody library comprising a plurality of antibodies, each antibody comprising at least six CDR regions (e.g., V H -CDRI, V H -CDR2, V H -CDR3, V L -CDRI, V L -CDR2, V L -CDR3).
• Each antibody in said partially diversified antibody library comprises certain “invariant CDR regions” and certain “variant CDR regions.”
• the term “invariant CDR regions” refers to CDR regions, which comprise the same nucleotide and amino acid sequence for every antibody in an antibody library and is derived from the parental or lead antibody.
• the term “variant CDR region” refers to a CDR region whose nucleotide and/or amino acid sequence is unique to an individual antibody or subset of antibodies within the antibody library.
• the partially diversified antibody library may comprise at least two invariant CDR regions (e.g., V H -CDR1, V H -CDR2, V H -CDR3, V L -CDR1, V L -CDR2, or V L - CDR3).
• the partially diversified antibody library comprises at least three, four or five invariant CDR regions. Limiting the number of variant CDR regions in a given antibody library decreases the combinatorial diversity between CDR regions and enables more variants at a single CDR region to be generated and screened. Maintaining two or more invariant CDR regions, also increases the chances of finding new variants that bind to the antigen in the same way, which is essential to retaining the biological activity of the parental antibody.
• the parental HCDR3 region is not varied.
• V L -CDR3, V H -CDR1, V H -CDR2, and V H -CDR3 are the same for each antibody of the plurality of antibodies of the antibody library.
• V L -CDR1, V L -CDR2, V H -CDR1, V H - CDR2, and V H -CDR3 are the same for each antibody of the plurality of antibodies of the antibody library.
• V L -CDR1, V L -CDR2, V L -CDR3, and V H -CDR3 are the same for each antibody of the plurality of antibodies of the antibody library.
• V L - CDR1, V L -CDR2, and V H -CDR3 are the same for each antibody of the plurality of antibodies of the antibody library.
• one of the two or more CDR sequences that are the same for each antibody of the plurality of antibodies of the antibody library is V H -CDR3.
• the CDR sequences are derived from a naturally occurring antibody library.
• only true natural CDRs are incorporated into the plurality of antibodies of the antibody library.
• One advantage of such an approach is that it avoids covariance violations, since the CDRs are known to fold correctly, as they have been derived from natural antibodies.
• the present disclosure thus features, in some embodiments, a method to create extremely diverse, highly functional antibody libraries by combining naturally occurring CDRs, including naturally occurring CDRs containing somatic mutations generated in vivo, within antibody scaffolds such that members of the antibody libraries are expected to be well expressed and/or folded, and lacking liabilities.
• the term “liability” refers to a motif in an antibody (e.g ., located in a heavy chain or light chain CDR region) that would negatively affect one or more desired features of the antibody (e.g., stability, good expression in an expression or display system, proper folding, no or reduced aggregation, solubility, no or reduced integrin binding, no or reduced glycosylation, no or reduced deamidation, no or reduced isomerization, no unpaired cysteine, or no or reduced protease sensitivity, etc.).
• desired features of the antibody e.g., stability, good expression in an expression or display system, proper folding, no or reduced aggregation, solubility, no or reduced integrin binding, no or reduced glycosylation, no or reduced deamidation, no or reduced isomerization, no unpaired cysteine, or no or reduced protease sensitivity, etc.
• an antibody library would be expected to be functionally much larger than libraries of similar genetic size, in which antibodies are present that contain any of
• the heavy chain (VH) and/or light chain (VL)- CDRl, CDR2, and/or CDR3 sequences for use in generating the antibody libraries described herein and for improving affinity maturation may be further analyzed to remove those that comprise a liability. Exemplary liabilities are listed in Table 1. In some embodiments, naturally occurring CDRs that contain any sequence liabilities are discarded and are not included in the antibody library.
• the CDRs (e.g CDR1, CDR2, CDR3, or a combination thereof) identified as described herein may be experimentally screened or selected for good folding and/or expression and screened or selected against liabilities such as poor folding, poor expression, polyreactivity or aggregation.
• the selected CDRs may be inserted into complete V domains within the context of the scaffolds.
• the resultant complete V domains could be further screened and selected for good folding and/or expression, and/or screened and selected against liabilities such as poor folding or expression, polyreactivity or aggregation.
• VH- and/or VL- CDR sequences comprising one of the liabilities listed in Table 1 (e.g ., a glycosylation site, a deamidation site, an isomerization site, an unpaired cysteine, a net charge greater than 1 (e.g., in LCDRl-2 and/or HC CDRl-2), a tripeptide motif containing at least two aromatic residues (which may affect viscosity), a motif that promotes aggregation, (viii) a poly specificity site such as those containing a motif of GG, GGG, RR, VG,
• glycosylation sites such as lysine glycolation sites may be removed.
• a glycolation site refers to a site in a protein molecule that can be linked to a sugar molecule via a non -enzymatic process.
• Exemplary glycolation sites include, but are not limited to, KE, EK, and ED.
• Additional liabilities include metal catalyzed fragmentation site (e.g., HS, SH, KT, HXS, or SXH, in which X represents any amino acid residue), poly specificity aggregation site (e.g., having a motif of X1X2X3, in which each of Xi, X2, and X3 is independently F, I, L, V, W, or Y), and streptavidin binding motif (e.g., HPQ, EPDW, PWXWL, in which X represents any amino acid residue, GDWVFI, and PWPWLG).
• metal catalyzed fragmentation site e.g., HS, SH, KT, HXS, or SXH, in which X represents any amino acid residue
• poly specificity aggregation site e.g., having a motif of X1X2X3, in which each of Xi, X2, and X3 is independently F, I, L, V, W, or Y
• Substantially free means that the number of a VH and/or VH CDR sequence comprising the liability is less than 20% in the library, e.g., less than 15% or less than 10%.
• VH and/or VL CDR1, CDR2, and/or CDR3 sequences comprising two or more (e.g., 3, 4, 5, 6, 7, or more) of the liabilities noted above can be removed such that the resultant library is free of (substantially free of or completely free of) members comprising the excluded liabilities.
• all of the liabilities listed in Table 1 can be removed such that the resultant library is free of (substantially free of or completely free of) members comprising any of the liabilities.
• the resultant heavy chain and/or light chain CDR1, CDR2, and/or CDR3 sequences obtained from naturally-occurring antibodies, either excluding sequences comprising one or more liabilities or maintaining all sequences, can be used as templates to synthesis nucleic acids encoding, and replicating, the CDR sequences for assembling the antibody library and use in the method described herein.
• nucleic acids can be inserted into the corresponding CDR position in the VH and/or VL chain of the lead antibody identified from the naive naturally occurring antibody library targeting an antigenic epitope.
• an initial antibody library may also be sorted for yeast displaying antibodies that have been stained with conformational probes that detect correct antibody folding.
• the sequences encoding functional members of the heavy and/or light CDR1, CDR2, and/or CDR3 can be used as templates for synthesizing nucleic acids coding for such functional members, or used directly.
• the resultant nucleic acids can then be inserted into the VH and/or VL chain of the lead antibody identified from the naive naturally occurring antibody library targeting an antigenic epitope as described herein to produce antibody libraries as also described herein.
• the antibody library disclosed herein is substantially free of non functional members, e.g., having less than 10% (e. g. , less than 8%, less than 5%, less than 3%, less than 1%, or lower) non-functional members.
• the methods for developing a high affinity antibody involves a two- phase diversification strategy that uses defined collections of natural CDRs purged of sequence liabilities.
• one or more antibody libraries i.e Partially Diversified Antibody Libraries
• the resulting antibodies are then selected for based on their binding affinity for the target and their epitope specificity (i.e., that they maintain the same epitope as the lead or parental antibody).
• the CDR regions of the selected antibody are combined to create a “combinatorial antibody library”, and the resulting antibodies in said combinatorial antibody are then selected for based on their binding affinities and epitope specificity as well.
• This two-step affinity maturation strategy allows the diversity at each CDR site to be explored more fully. Library size is often a concern when performing in vitro evolution of any sort since one is limited by the number of transformants that can be conveniently obtained during library generation. Varying only a subset of the CDR regions in each initial partially diversified antibody libraries, allows one to explore the sequence space more effectively, while retaining epitope specificity for the target. As disclosed herein, this methodology results in affinity improvement of an antibody lead to low-picomolar KD values.
• the invention relates to a method of generating a combinatorial antibody library, comprising, generating at least two antibody libraries that each comprises a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises: a V H -CDR1 sequence, a V H -CDR2 sequence, a V H -CDR3 sequence; a V L -CDR1 sequence, a V L -CDR2 sequence, and a V L -CDR3 sequence, wherein each antibody library is generated by selecting two or more CDR sequences as invariant CDR sequences, wherein said invariant CDR sequences are derived from a parental antibody known to bind to the specified target antigen; generating an antibody library comprising a plurality of antibodies, wherein each antibody of the plurality of antibodies comprises: the two or more invariant CDR sequences selected; and a unique combination of variant CDR sequences not selected in; wherein the CDR sequences are derived from the sequences of CDRS found in naturally occurring antibodies; screening
• the combinatorial library is generated by combining the CDR regions of selected high affinity antibodies from at least two partially diversified antibody libraries. In some embodiments the combinatorial library is generated by combining the CDR regions of selected high affinity antibodies selected from at least two, three, four, five, six, seven, eight, nine or ten partially diversified antibody libraries. In some embodiments, each of the partially diversified antibody libraries comprises one of the following sets of invariant CDR regions:
• each of the partially diversified antibody libraries comprises an invariant V H -CDR3 domain.
• the combinatorial antibody library may not contain any invariant CDR regions.
• the combinatorial antibody will contain one or more invariant CDR regions.
• the combinatorial antibody will comprise one, two, three, four or five invariant CDR regions.
• the combinatorial antibody library may comprise an invariant VH- CDR3 domain.
• the combinatorial antibody library can be of any form.
• the antibody library is a full-length antibody library, a Fab antibody library, a single chain antibody library, or a single domain antibody library.
• the antibody library is a human antibody library. It is not necessary that the combinatorial antibody library be of the same type of antibody library as the partially diversified antibody libraries.
• the combinatorial antibody library might be a full-length antibody library, and the partially diversified antibody libraries used to generate the selected antibodies whose CDR regions make up the combinatorial library may be single chain antibody libraries.
• the selected one or more CDR sequences that are the same for each antibody of the plurality of antibodies for the methods and antibody libraries may be (V L -CDR3,V H -CDR1, V H -CDR2, and V H -CDR3) [for the L1L2 library], (V L -CDR1, V L -CDR2, V H -CDR1, V H -CDR2, and V H- CDR3) [for the L3 library], (V L- CDR1, V L -CDR2, V L -CDR3, and V H -CDR3) [for the H1H2 library], (V H -CDR3) [for the Combo 1 library], and (V L -CDRI, V L -CDR2, and V H -CDR3) [for the Combo 2 library].
• the CDR sequences are derived from a naturally occurring antibody library wherein the antibodies therein bind a single antigen or a single antigenic epitope and the binding affinity is at least 9.5 nM.
• the selected one or more CDR sequences form the invariant CDRs in the assembled combinatorial antibody.
• the remaining non-selected CDRs that vary for each antibody of the plurality of antibodies may be (V L -CDRI and V L -CDR2) [for the L1L2 library], (V L -CDR3) [for the L3 library], (V H -CDR1 and V H -CDR2) [for the H1H2 library], (V L - CDRI, V L -CDR2, V L -CDR3, V H -CDR1, V H -CRD2) [for the Combo 1 library], and (V L -CDR3, V H -CDR1, V H -CDR2) [for the Combo 2 library].
• the CDR sequences are free of one or more of the following sequence liabilities: (i) a glycosylation site, (ii) a deamidation site, (iii) an isomerization site, (iv) unpaired cysteine, (v) net charge greater than 1 , (vi) a tripeptide motif containing at least two aromatic residues, (vii) a motif that promotes aggregation, (viii) a poly specificity site; (ix) a protease sensitive site, (x) an integrin binding site, (xi) a lysine glycosylation site, (xii) a metal catalyzed fragmentation site, (xiii) a poly specificity aggregation site; and (xiv) a streptavidin binding motif.
• the glycosylation site may comprise the motif NXS, NXT, or NXC, in which X represents any naturally-occurring amino acid residue except for proline;
• the deamidation site may comprise the motif of NG, NS, NT, NN, NA, NH, ND, GNF, GNY, GNT, or GNG;
• the isomerization site may comprise the motif of DT, DH, DS, DG, or DD; tripeptide may be HYF or HWH;
• the motif that promotes aggregation may comprise the motif of FHW;
• the poly specificity site may comprise the motif GG, GGG, RR, VG, W, WV, WW, WWW, YY, or WXW, in which X represents any amino acid residue;
• the protease cleavage site may comprise the motif of DX, in which X is P, G, S, V, Y, F, Q, K, L, or D;
• the antibody libraries disclosed herein can be made using known techniques in the art. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Floogenboom et al in Methods in Molecular Biology 178:1-37 (O'Brien et al, ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al, Nature 348:552-554; Clackson et al, Nature 352: 624-628 (1991); Marks et al, J. Mol. Biol.
• any of the antibody libraries described herein may be used to screen for antibodies having binding specificity to an antigen of interest.
• Antibodies encoded by the nucleic acids in the library can be expressed and displayed using a suitable expressing/display system, for example, a cell-free display system (e.g., ribosome display), a phage display system, a prokaryotic cell-based display system (e.g ., bacterial display), or a eukaryotic cell-based display system (e.g., yeast display or mammalian cell display).
• the antibody libraries are expressed and displayed on yeast cells.
• the antibody libraries are expressed and displayed on phage particles (phage display).
• two or more display systems are used, e.g., phage display followed by yeast display.
• the library of antibodies may be expressed/displayed in a suitable system, e.g., those described herein, in any format. Examples include intact antibodies (full-length antibodies), antigen-binding fragments thereof (e.g., Fab), or single chain antibodies (scFv or scFab).
• Phage display is a protein display format using bacteriophages (e.g., phage fl, fd, and M13).
• at least one antibody chain e.g., the heavy chain and/or the light chain
• a bacteriophage coat protein for example, a gene III protein, a gene VIII protein, or a major coat protein (see, e.g., WO 00/71694).
• Phage display is described, for example, in U.S. 5,223,409; Smith (1985) Science 228: 1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haard etal. (1999) J Biol. Chem 274: 18218-30; Hoogenboom etal. (1998) Immunotechnology 4: 1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8; Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay etal.
• Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g., PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected protein components can be isolated from cells infected with the selected phages or from the phage themselves, after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.
• a eukaryotic expression/display system e.g., yeast cells or mammalian cells
• yeast cells can be used for expressing and displaying the library of antibodies as described herein.
• Yeast display is a protein display format, in which a protein component (e.g., an antibody component) is linked to a yeast cell wall protein (e.g., Agalp or Aga2p) directly or indirectly.
• a protein component e.g., an antibody component
• yeast cell wall protein e.g., Agalp or Aga2p
• one chain of an antibody can be covalently fused to the yeast cell wall protein for direct display.
• the association between an antibody component and a yeast cell wall component can be mediated by an intermediate agent.
• Yeast display is described in, e.g., Cho et al, J. Immunol.
• Phage particles or host cells displaying antibodies binding to the target antigen can be isolated, for example, by retention or a support member on which the target antigen is immobilized, amplified if needed, and the nucleic acids coding for the displayed antibodies can be determined.
• the screening process can be repeated multiple time, and display systems can be used in combination. When needed different antigens can be used for selecting antibody members having desired binding specificity or for negative selection to exclude antibody members having binding activity to a non-target antigen.
• binding activity can be evaluated by standard immunoassay and/or affinity chromatography. Determining the ability of candidate antibodies to bind therapeutic targets can be assayed in vitro using, e.g., a BIACORETM instrument, which measures binding rates of an antibody to a given target antigen based on surface plasmon resonance. In vivo assays can be conducted using any of a number of animal models and then subsequently tested, as appropriate, in humans. Cell-based biological assays are also contemplated.
• Affinity maturation is a necessary step for the development of potent therapeutic molecules.
• Technology for introducing diversity into proteins has long existed and has been extensively used to evolve molecules with desired characteristics, including affinity maturation of antibodies in vitro (Kang, Jones et al. 1991, Hawkins, Russell et al. 1992, Marks, Griffiths et al. 1992, Jackson, Sathe et al. 1995, Yang, Green et al. 1995, Low, Holliger et al. 1996, Schier and Marks 1996, Schier, McCall et al. 1996, Thompson, Pope et al. 1996, Hanes, Jermutus et al. 1998, Hemminki, Niemi et al. 1998, Wu, Beuerlein et al.
• the present disclosure demonstrates the affinity improvement of lead antibodies to low- picomolar KD values, while retaining identical epitope binding, and with the reduction of the number of sequence liabilities. This is based on the use of a new diversification strategy that uses defined collections of natural CDRs purged of sequence liabilities.
• CDR sequences were identified by NGS of a naive library (Erasmus, D’Angelo et al. 2021, see, U.S. Patent No. 10,954,508, the content of which is hereby incorporated by reference in its entirety).
• the antibody against a monomeric human protein of therapeutic interest was chosen to be affinity matured using the improved affinity maturation method.
• the antibody comprising a VH3 heavy chain and a VA3 light chain, was initially identified by biopanning a naive human scFv phage display library (Erasmus, D’Angelo et al. 2021).
• Analysis of the CDRs revealed three sequence liabilities with the potential of affecting downstream clinical development: two distinct aspartate isomerization sites, which can lead to chemical degradation and loss of potency (Sydow, Lipsmeier et al. 2014), at LCDR2 and LCDR3, and a GG non-specificity motif (Kelly, Le et al. 2018) at HCDR2.
• N-linked NxS and NxT where x is not a Gavel and von glycosylation Pro Heijne 1990; Kumar and Singh 2016
• Library size is often a concern when performing in vitro evolution of any sort since one is limited by the number of transformants that can be conveniently obtained during library generation: 10 9 -10 10 for phage and E. coli display and 10 8 -10 9 for yeast display in S. cerevisiae.
• the present disclosure opted for a step-wise approach (Hemminki, Niemi et al. 1998) since it would allow exploring the sequence space more effectively: in phase 1, LCDR1 was combined with LCDR2, HCDR1 and HCDR2, and LCDR3 was left by itself (Table 3), but there was no reason to believe that this was somehow the optimal approach. It was done so out of what seemed to be the most convenient combinations for library building.
• each library allowed generation of more transformants than the maximum combinatorial diversities for each (theoretical diversity ranging from 1.7xl0 5 to 3.2xl0 7 ). Also, by fixing at least four parental CDRs, including the all-important HCDR3, in each library, search space was decreased, increasing the chances of finding new variants binding to the antigen in the same way, essential to retaining biological activity.
• each library also included the parental CDR sequences at the same abundance as the other introduced CDRs, even if they contained sequence liabilities. This was to ensure retained activity in the case that particular parental CDR sequences were essential for binding.
• CDR1 The five different collections of CDR sequences (LCDR1, LCDR2, LCDR3, HCDR1, and HCDR2) were amplified with specific primers by PCR using Q5 polymerase (NEB #M0491L). The remaining regions were amplified from the parental scFv and assembled with the CDRs by PCR (see, FIG.s 1A and 1C). Libraries were assembled by combining the newly produced CDR pools with the remaining parts of the scFv.
• the L1L2 library was assembled by (1) amplifying the LCDR1 and LCDR2 with the flanking frameworks from the synthetic oligo pool, (2) amplifying the remaining parts of the scFv from the parental clones, (3) assembling the produced fragments by overlap PCR, and (4) transforming the produced scFv cassettes into S. cerevisiae along with the digested yeast display vector (FIG. 1C).
• the scFv amplicons from each library were transformed into yeast along with the yeast display vector pSYD previously digested with the enzymes BssFIII and Nhel (NEB #R0199S and #R0131S) by electroporation using method described previously (Benatuil, Perez et al. 2010).
• the present disclosure chose to use scFv yeast display because it provides high precision in retrieving the desired population when combined with flow cytometry. Flowever, it was expected this diversification approach was equally effective in the phage or ribosome display context and one could even expand possibilities due to larger library sizes. However, experimental design during selection would have to be adapted to these platforms since they are expected to show different behavior due to their monovalent nature as opposed to the multivalent nature of yeast: whereas a polyvalent yeast cell exists in a continuum from antigen saturation to no antigen binding that varies over time in proportion to the antibody off-rate, monovalent systems can only exist in the binary bound or not-bound states.
• This difference can be overcome, for example, by using a larger number of displaying particles and relying on the population binding decay over time as opposed to the single cell decay.
• the present disclosure routinely use the scFv format for antibody discovery and engineering and have found conversion to the IgG format occurs with 70-90% success. However, if conversion is a concern, the techniques described here would be easily applied using a Fab display system.
• Yeast display selections were performed as in Ferrara et al (2012). Briefly, cells were induced in selective media containing 2% galactose overnight at 20°C. 10 5 induced cells are washed twice with cold washing buffer (PBS pH 7.40.5% BSA) and incubated at room temperature with the biotinylated antigen diluted in PBS. Two different selection strategies were used in this phase: equilibrium selection and kinetic selection (FIGS. 2A-2B) (Boder and Wittrup 1998, Boder, Midelfort et al. 2000).
• Equilibrium selection the more traditional approach, was performed by incubating the scFv-displaying yeast cells with a defined concentration of labeled antigen (biotinylated, in this case) and sorting labeled cells immediately after reaching equilibrium. Incubations are often performed with decreasing antigen concentrations as the selections round progress.
• the cells are washed and stained promptly with the anti-SV5 labeled with PE (phycoerythrin; labels cells displaying scFv) and streptavidin labeled with Alexa Fluor 633 (Thermo Scientific; labels cells bound to biotinylated antigen) and then cells binding the antigen are sorted either by FACS (fluorescence-activated cell sorting) or MACS (magnetic-activated cell sorting).
• FACS fluorescence-activated cell sorting
• MACS magnetic-activated cell sorting
• the present disclosure performed an initial flow cytometric assessment of the libraries using decreasing antigen concentrations (FIG. 2C).
• the light chain libraries, L1L2 and L3, showed a small population binding to the antigen even at the highest concentration used: at 100 nM binding populations of 3% and 1.5% respectively.
• the heavy chain library, H1H2 significant binding could be observed from 1.2 nM (2.9% of the population) to 100 nM (8.3% of the population), suggesting higher improvement potential for the heavy chain CDRs as opposed to the light chains for this particular clone.
• the present disclosure performed first and second rounds of selection using magnetic-assisted cell sorting (MACS) at antigen concentrations of 10 nM and 1 nM, respectively. This allowed the present disclosure to label and sort a larger number of cells than what would be practical using a flow cytometer.
• the present disclosure used fluorescence activated cell sorting (FACS) to enable more precise sorting of the cells of interest.
• FACS fluorescence activated cell sorting
• the present disclosure combined the selection outputs with the goal to further improve binding towards still slower off-rates.
• Two “combo” libraries were assembled by PCR: the first (Combo 1 - 2.27x10 8 transformants) was created by combining the output of all three libraries (L1L2, L3, and H1H2), while the second (Combo 2 - 1.04x10 s transformants) omitted the L1L2 output and used the parental CDRs at LCDR1 and LCDR2 (FIG. 3A). Again, an approach combining equilibrium and kinetic sorts was used (FIG. 3B) with two successive rounds of kinetic sorting at 4h and 16h with unlabeled antigen respectively, followed by a final equilibrium round at 0.1 nM antigen concentration.
• the present disclosure converted the populations obtained after the 3rd round of selection of the Combo libraries to an scFv-Fc format to facilitate affinity screening.
• the scFv from the final population (round 3, combo libraries) was bulk cloned into a yeast expression vector containing a human IgGl Fc region to be expressed in the scFv-Fc format.
• the scFv region was amplified by PCR, digested with BssFlII and Nhel restriction enzyme (New England Biolabs) and cloned into the pDNL9 vector.
• HCDR1 and HCDR2 As for HCDR1 and HCDR2, much of the diversity found is concentrated in a few positions/hotspots, suggesting that these may be less relevant for binding (e.g.: position 6 at both HCDR1 at HCDR2), while some other positions showed clear convergence to an amino acid different to the parental (FIG. 4C, dashed squares). Additionally, two of three isomerization motifs may be eliminated, and the GG polyreactivity motif may be eliminated, resulting in the potential elimination of three of four sequence liabilities.
• Removing liabilities from a therapeutic mAb has a series of benefits: de-risking the lead upfront can improve developability and minimize chances of clinical failure due to poor molecule properties (Jain, Sun et al. 2017).
• increased immunogenicity risk e.g., glycosylation
• loss of potency e.g., asparagine deamidation
• CQA Critical Quality Attributes
• scFv-Fc were expressed as scFv-Fc in S. cerevisiae yeast strain YVH10 (ATCC MYA-4940): 11 from the Combo 1 library (A01-A06, B01-B06) and 12 from the Combo 2 library (A07-A12, B07-B12). These had 3 to 13 amino acid changes from the parental. To determine whether they retained the same epitope as the initial lead, it was tested if these were able to inhibit the parental scFv from binding to the antigen. The present disclosure incubated the scFv supernatants with the labeled antigen (10 nM) for 15 minutes.
• clones are often tested for their ability to achieve the desired biological activity.
• the epitope recognized by the antibody will dictate if the antibody will be an agonist, antagonist, or have no activity whatsoever.
• leads with desired activity it is assumed that increasing affinity will also increase potency (Rosenfeld et al. 2017; Hurlburt et al. 2020), and retaining the recognition site is one of the basic requirements of this assumption.
• More aggressive maturation techniques such as chain shuffling (especially heavy chain, or portions thereof) may have a higher potential to cause epitope drift and loss of activity, making a previously valuable molecule useless.
• the present disclosure has shown that a stepwise approach where at least 4 CDRs remain constant, and very importantly, the HCDR3 remains unchanged, was effective in obtaining very high affinities while retaining recognition of the same epitope. Whereas every antibody-antigen pair had its own peculiarities, it was believed that the centrality of the HCDR3 in epitope recognition (Xu and Davis 2000, Akbar, Robert et al. 2021) justified the retention of the parental HCDR3 to avoid epitope drift.
• the present disclosure determined the affinity of the same 23 clones using high- throughput SPR.
• the Carterra LSA surface resonance system was used for the affinity measurements. Briefly, anti-Human IgG Fc (Southern Biotech, #2048-01) was chemically coupled to an HC30M chip following manufactures protocols. Crude yeast supernatants containing the scFv-Fc fusions were arrayed on the chip. Non-biotinylated antigen was injected at varying concentrations (0.08 nM to 50 nM) to determine association and dissociation rates. All analyses were performed using Carterra software and sensorgrams were fitted using a pseudo first order kinetic model (Lundstrom 1994).
• Example 8 Parental Antibody Identification and CDR Design Including HCDR3
• HCDR3 it may be desirable to introduce limited diversity into the HCDR3 as well.
• HCDR3 is considered the most important CDR mediating antibody binding specificity
• extensive mutation in the HCDR3 may be detrimental, in that binding specificity of the antibody under examination may be modified.
• An antibody against a monomeric human protein of therapeutic interest was chosen to be affinity matured using the improved affinity maturation method.
• the antibody comprises a VH3 heavy chain and a VK1-39 light chain.
• Analysis of the CDRs revealed three sequence liabilities with the potential of affecting downstream clinical development: two distinct YY polyreactivity sites, which can lead to aggregation and non specificity at HCDR3 and LCDR3, and one hydrophobic site at LCR2.
• the scFv affinity was determined by yeast display flow cytometry. Briefly, cells were washed twice with cold washing buffer (PBS pH 7.40.5% BSA) and incubated at room temperature with different concentrations of biotinylated antigen, from 4 nM to 3 mM. After the biotinylated antigen incubation step the cells are washed and stained with the anti-SV5 labeled with PE (phycoerythrin; labels cells displaying scFv) and streptavidin labeled with Alexa Fluor 633 (Thermo Scientific; labels cells bound to biotinylated antigen) and then cells are analyzed by flow cytometry.
• PE phytoerythrin
• Alexa Fluor 633 Thermo Scientific; labels cells bound to biotinylated antigen
• the present disclosure used deep sequencing data (MiSeq and NovaSeq) from a naive semisynthetic library (Azevedo Reis Teixeira, Andre et al. 2021, Bradbury, A.R.M. et al. 2020).
• CDRs from B cell receptor antibodies are embedded within well-behaved clinical antibody scaffolds, the HCDR3s, due to their high diversity, are directly amplified from B cell mRNA, and the remaining naturally replicated CDRs are identified from next generation sequencing (NGS) of numerous donors and synthesized on arrays after eliminating previously identified sequence liabilities.
• NGS next generation sequencing
• LCDRl-3 only CDR sequences coming from the VK1 germline family were considered and for HCDRl-2 only sequences coming from the VH3 family were considered, assuming that these sequences, since they belonged to the same germline genes as the parental antibody, would be better tolerated and minimize any potential structural disruption.
• N-linked NxS and NxT where x is not a Gavel and von glycosylation Pro Heijne 1990; Kumar and Singh 2016
• each amino acid position was mutated to 19 amino acids (excluding cysteine) for a total of Y x 19 oligonucleotides, where Y is the number of HCDR3 amino acids to be mutated.
• the present disclosure believe the centrality of the HCDR3 in epitope recognition (Xu and Davis 2000, Akbar, Robert et al. 2021) justifies the application of the mutational scanning approach to identify mutations that are better tolerated and to simultaneously avoid epitope drift, minimizing any potential structural disruption.
• the identified CDRs were generated with flanking framework sequences matching the parental antibody and produced using array-based DNA synthesis (Agilent). This allowed the rescue of the full diversity at each individual CDR site by using framework primers.
• Library size is often a concern when performing in vitro evolution of any sort since one is limited by the number of transformants that can be conveniently obtained during library generation: 10 9 -10 10 for phage and E. coli display and 10 8 -10 9 for yeast display in S. cerevisiae.
• the present disclosure opted for a step-wise approach (Hemminki, Niemi et al. 1998) since it would allow exploring the sequence space more effectively: in phase 1, HCDR1 was combined with HCDR2, LCDR1 and LCDR2, and leave HCDR3 and LCDR3 by themselves (Table 6), but there was no reason to believe that this was somehow the optimal approach. It was done so out of what seemed to be the most convenient combinations for library building.
• each library also included the parental CDR sequences at the same abundance as the other introduced CDRs, even if they contained sequence liabilities. This was to ensure retained activity in the case that particular parental CDR sequences were essential for binding.
• Table 6 shows number of different sequences introduced in each CDR position of light and heavy chain of four Phase I libraries. Theoretical diversity is calculated by the combinatorial potential of the CDRs and reported number of transformants correspond to the yeast display libraries created.
• the six different collections of CDR sequences (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) were amplified with specific primers by PCR using Q5 polymerase (NEB #M0491L). The remaining regions were amplified from the parental scFv and assembled with the CDRs by PCR ⁇ see, FIGS. 7A-7B). Libraries were assembled by combining the newly produced CDR pools with the remaining parts of the scFv.
• the H1H2 library was assembled by (1) amplifying the HCDR1 and HCDR2 with the flanking frameworks from the synthetic oligo pool, (2) amplifying the remaining parts of the scFv from the parental clones, (3) assembling the produced fragments by overlap PCR, and (4) transforming the produced scFv cassettes into S. cerevisiae along with the digested yeast display vector (FIG. 7B).
• the scFv amplicons from each library were transformed into yeast along with the yeast display vector pSYD previously digested with the enzymes BssHII and Nhel (NEB #R0199S and #R0131S) by electroporation using method described previously (Benatuil, Perez et al. 2010).
• the present disclosure chose to use scFv yeast display because it provides high precision in retrieving the desired population when combined with flow cytometry.
• this diversification approach was equally effective in the phage or ribosome display context, or any other display platform, and one could even expand possibilities due to larger library sizes.
• experimental design during selection would have to be adapted to these platforms since they are expected to show different behavior due to their monovalent nature as opposed to the multivalent nature of yeast: whereas a polyvalent yeast cell exists in a continuum from antigen saturation to no antigen binding that varies over time in proportion to the antibody off-rate, monovalent systems can only exist in the binary bound or not-bound states.
• This difference can be overcome, for example, by using a larger number of displaying particles and relying on the population binding decay over time as opposed to the single cell decay.
• the present disclosure routinely use the scFv format for antibody discovery and engineering and have found conversion to the IgG format occurs with 70-90% success. However, if conversion is a concern, the techniques described here would be easily applied using a Fab display system.
• Yeast display selections were performed as in Ferrara et al (2012). Briefly, cells were induced in selective media containing 2% galactose overnight at 20°C. 10 5 induced cells are washed twice with cold washing buffer (PBS pH 7.40.5% BSA) and incubated at room temperature with the biotinylated antigen diluted in PBS. Equilibrium selection was performed by incubating the scFv-displaying yeast cells with decreasing concentrations of biotinylated antigen as the selections round progress and sorting labeled cells immediately after reaching equilibrium (FIG. 2A).
• the cells are washed and stained promptly with the anti-SV5 labeled with PE (phycoerythrin; labels cells displaying scFv) and streptavidin labeled with Alexa Fluor 633 (Thermo Scientific; labels cells bound to biotinylated antigen) and then cells binding the antigen are sorted either by FACS (fluorescence- activated cell sorting) or MACS (magnetic-activated cell sorting) (FIG. 2A).
• FACS fluorescence- activated cell sorting
• MACS magnetic-activated cell sorting
• the present disclosure performed an initial flow cytometric assessment of the libraries using decreasing antigen concentrations (FIG. 8A).
• the light chain library L1L2 showed a reduced population binding to the antigen even at the highest concentration used: at 400 nM binding population of 0.031%.
• Libraries H1H2 and L3, showed a small population binding to the antigen at 400 nM: binding populations of 0.63% and 0.2% respectively.
• the heavy chain library H3 a significant population of 6.81% binding to the antigen even at 100 nM was identified, suggesting higher improvement potential for the heavy chain CDRs as opposed to the light chains for this particular clone.
• the present disclosure performed the first round of selection using magnetic-assisted cell sorting (MACS) at antigen concentration of 400 nM (FIG. 8B). This allowed the present disclosure to label and sort a larger number of cells than what would be practical using a flow cytometer.
• the present disclosure used fluorescence activated cell sorting (FACS) to enable more precise sorting of the cells of interest (FIG. 8B).
• FACS fluorescence activated cell sorting
• FIG. 8C An assessment of the population obtained for each of these libraries after three rounds show that in all cases a significant improvement of affinity can be observed: all generated populations show significant binding to the antigen even at 10 nM, a concentration at which the parental antibody does not bind. Even though affinities of individual clones have not been assessed at this stage, it is not unreasonable to assume that, given the yeast display staining profile, one could already find binders with satisfactory affinities depending on the requirements of the project.
• the present disclosure combined the selection outputs with the goal to further improve binding towards still slower off-rates.
• Two “combo” libraries were assembled by PCR: the first (Combo 1 - 6.8E+08 transformants) was created by combining the output of all four libraries (H1H2, H3, L1L2 and L3), while the second (Combo 2 - 6.5E+08 transformants) omitted the H3 output and used the parental HCDR3 (FIG. 3A and FIG. 9A).
• scFv-displaying yeast cells are incubated with the labeled antigen, washed, incubated with unlabeled antigen (lOx more concentrated than the biotinylated antigen) to select only clones with stable binding to the antigen (slow off-rate - k d ).
• the unlabeled antigen is used to prevent rebinding of the displaced labeled antigen. After a defined period, cells still bound to the labeled antigen were stained and sorted as described before.
• Combo library 1 identified clones had high tolerance for diversity on HCDR3, however, most clones retained the second YY polyreactivity site and were mutated at a different position. Nevertheless, 11 clones derived from Combo library 1 were free of liabilities at HCDR3. In summary, 11 affinity matured clones retained a single CDR liability (hydrophobic site at LCR2), 40 clones had two sequence liabilities (hydrophobic site at LCR2 and YY polyreactivity site at HCDR3), while the parental clone had 3 sequence liabilities, showing that the method can be effectively used to reduce developability issues simultaneously with affinity maturation.
• Thermo Scientific labels cells bound to biotinylated antigen
• the mean affinity for clones selected from Combo 1 library was 5.609+0.4931 nM, ranging from 3.879 to 9.927 nM.
• mean affinity was 8.075+1.758 nM, ranging from 4.215 to 24.84 nM (Table 8).
• inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
• inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
• a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
• the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
• This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
• “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
• the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

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